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SLAMseq

High-Throughput Kinetic RNA Sequencing

Getting Started with SLAMseq Explorer Kits

SLAMseq uses the ribonucleoside analog 4-thiouridine (S4U) to label newly synthesized (nascent) RNA in living cells. Labeling abilities deviate between cultured cells depending on cell type, culturing conditions, media compositions, cell viability, or cell physiology.

Optimize 4-Thiouridine (S4U) Labeling Conditions to Maximize Impact

For all new setups, SLAMseq requires cell type- and setup-specific optimization of labeling conditions and efficiency testing of S4U uptake (exploring phase) prior to kinetics experiment (Fig. 1). Once optimized, the exploring phase can be skipped for subsequent experiments with the same cell type and experimental settings. Initial validation and further optimization are highly recommended when relying on published, cell-type specific S4U working concentrations.

Optimal S4U working concentrations are determined by assessing cell viability over time in a simple titration experiment. The efficiency of S4U uptake into the nucleus is measured by shallow sequencing (recommend) or HPLC.

Get in touch with our specialists while you are planning your SLAMseq experiment!
For more information and useful tips, contact us at support@lexogen.com.

Figure 1 | Overview of SLAMseq workflows for high-throughput kinetic RNA sequencing (*Optimization of labeling conditions during the exploring phase is highly recommended when working with SLAMseq for the first time, using a different cell type, with changed parameters of experimental setup, or for adjusting published labeling references.).

Typical Results

With the SLAMseq Explorer Kits for 3’ mRNA-Seq with QuantSeq FWD V2, labeling concentrations are optimized towards cell type and experimental setup and S4U incorporation is effectively checked by shallow sequencing. The S4U concentration cytotoxicity is measured over a time scale. The inhibition vs S4U concentration curve is determined by measuring cell viability over an S4U dilution series (Fig. 2). The optimal experimental working concentration is defined as the S4U concentration (= IC10,ti) that would inhibit a maximum of 10 % of cells in the given time window (ti).

Figure 2 | Assessing optimal S4U working concentration at IC10 in titration experiment by plotting various S4U concentration against cell viability. Sigmoidal curve fitting is used to determine the half-maximal inhibitory concentration, IC50,ti.

With the SLAMseq Explorer Kits for HPLC, labeling concentrations are optimized towards cell type and experimental setup and S4U incorporation is checked by HPLC. The optimal experimental working concentration is defined as the S4U concentration (= IC10,ti) that would inhibit a maximum of 10 % of cells in the given time window (ti).

Figure 3 | Incorporation rate of S4U as a percentage of total uridine levels, as determined by HPLC.

FAQ

Frequently Asked Questions

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Please also check our General Guidelines and FAQ resources!

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Safety Data Sheet

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

Ordering Information

Cat. №Product Name
059.24SLAMseq Explorer Kit - Cell Viability Titration Module, 24 preps
060.24SLAMseq Explorer Kit - S4U Incorporation Module, 24 preps
227.24SLAMseq Explorer Kits for 3’ mRNA-Seq with QuantSeq FWD V2, 24 preps
228.24SLAMseq Explorer Kits for using with HPLC, 24 preps

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Try the SLAMseq product configurator and find the ideal setup for your application!

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